HPLC-based activity profiling approach for the discovery of GABAA receptor ligands using an automated two microelectrode voltage clamp assay on Xenopus oocytes.

An approach for rapid HPLC-based profiling for new GABA (A) ligands of natural origin has been developed. Active extracts are separated by a single injection of 3-10 mg of extract onto a semi-preparative (150 x 10 mm i. d.) HPLC column with gradient elution and time-based fractionation. The microfractions are tested in an automated two-microelectrode voltage-clamp assay on Xenopus oocytes expressing recombinant GABA (A) channels composed of alpha (1), beta (2) and gamma (2S) subunits. The protocol has been validated by spiking experiments with inactive extract and the GABA (A) receptor ligand magnolol, and by profiling of active extracts such as valerian extract containing the known GABA (A) receptor ligand valerenic acid. For dereplication of GABA containing extracts, we established a rapid and simple procedure by which GABA is analyzed as OPA derivative by reversed-phase HPLC. This dereplication protocol was validated with plant and fungal extracts which had been previously tested active or inactive in the oocyte assay and with spiking experiments.

[1]  M. Melzig,et al.  HPLC Based Activity Profiling for Inhibitors of Human Neutrophil Elastase in Isatis tinctoria Leaf Extracts , 2006 .

[2]  M. Hamburger,et al.  Identification and isolation of the cyclooxygenase-2 inhibitory principle in Isatis tinctoria. , 2001, Planta medica.

[3]  H. Xue,et al.  Naturally occurring 2'-hydroxyl-substituted flavonoids as high-affinity benzodiazepine site ligands. , 2003, Biochemical pharmacology.

[4]  F. Stephenson.  THE GABA(A) RECEPTORS , 1995 .

[5]  J. Medina,et al.  Benzodiazepine-like molecules, as well as other ligands for the brain benzodiazepine receptors, are relatively common constituents of plants. , 1989, Biochemical and biophysical research communications.

[6]  O. Sterner,et al.  Honokiol and Magnolol Increase the Number of [3H]Muscimol Binding Sites Three-Fold in Rat Forebrain Membranes In Vitro Using a Filtration Assay, by Allosterically Increasing the Affinities of Low-Affinity Sites , 1999, Neurochemical Research.

[7]  J. Casida,et al.  Alpha-thujone (the active component of absinthe): gamma-aminobutyric acid type A receptor modulation and metabolic detoxification. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[8]  Josef Hölzl,et al.  Lignans isolated from valerian: identification and characterization of a new olivil derivative with partial agonistic activity at A(1) adenosine receptors. , 2002, Journal of natural products.

[9]  W. Sieghart,et al.  Pharmacological Properties of GABA A Receptors Containing 1 Subunits □ S , 2006 .

[10]  Chun-Su Yuan,et al.  The Gamma-Aminobutyric Acidergic Effects of Valerian and Valerenic Acid on Rat Brainstem Neuronal Activity , 2004, Anesthesia and analgesia.

[11]  W. Hevers,et al.  The diversity of GABAA receptors , 1998, Molecular Neurobiology.

[12]  P. Whiting.  GABA-A receptors: a viable target for novel anxiolytics? , 2006, Current opinion in pharmacology.

[13]  Sabrina Moret,et al.  HPLC determination of free nitrogenous compounds of Centaurea solstitialis (Asteraceae), the cause of equine nigropallidal encephalomalacia. , 2005, Toxicon : official journal of the International Society on Toxinology.

[14]  W. Sieghart,et al.  Pharmacological Properties of GABAA Receptors Containing γ1 Subunits , 2006, Molecular Pharmacology.

[15]  I. Baburin,et al.  Automated fast perfusion of Xenopus oocytes for drug screening , 2006, Pflügers Archiv.

[16]  H. Xue,et al.  Development of effective therapeutics targeting the GABAA receptor: naturally occurring alternatives. , 2004, Current pharmaceutical design.

[17]  I. Khan,et al.  Chemical fingerprinting of valeriana species: simultaneous determination of valerenic acids, flavonoids, and phenylpropanoids using liquid chromatography with ultraviolet detection. , 2006, Journal of AOAC International.

[18]  I. Paulsen,et al.  Determinants of amentoflavone interaction at the GABA(A) receptor. , 2005, European journal of pharmacology.

[19]  A. Trzeciak,et al.  Distribution, prevalence, and drug binding profile of gamma-aminobutyric acid type A receptor subtypes differing in the beta-subunit variant. , 1994, The Journal of biological chemistry.

[20]  A. Trzeciak,et al.  GABAA receptors display association of gamma 2-subunit with alpha 1- and beta 2/3-subunits. , 1991, The Journal of biological chemistry.

[21]  K. Dekermendjian,et al.  6‐Methylflavone, a benzodiazepine receptor ligand with antagonistic properties on rat brain and human recombinant GABAA receptors in vitro , 1997 .

[22]  Matthias Hamburger,et al.  Natural products in drug discovery –concepts and approaches for tracking bioactivity , 2006 .

[23]  F. Stephenson,et al.  The GABAA receptors. , 1995, The Biochemical journal.

[24]  E. Sigel,et al.  Novel Plant Substances Acting as β Subunit Isoform-Selective Positive Allosteric Modulators of GABAA Receptors , 2005, Molecular Pharmacology.

[25]  B. Sakmann,et al.  Patch clamp measurements onXenopus laevis oocytes: currents through endogenous channels and implanted acetylcholine receptor and sodium channels , 1986, Pflügers Archiv.

[26]  R. Mckernan,et al.  theta, a novel gamma-aminobutyric acid type A receptor subunit. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[27]  S. Hering.  Small-volume and rapid extracellular solution exchange around Xenopus oocytes during voltage-clamp recordings , 1998, Pflügers Archiv.

[28]  I. Baburin,et al.  Valerenic acid potentiates and inhibits GABAA receptors: Molecular mechanism and subunit specificity , 2007, Neuropharmacology.

[29]  W. Stigelman,et al.  Goodman and Gilman's the Pharmacological Basis of Therapeutics , 1986 .

[30]  C. Gerhäuser,et al.  HPLC-based activity profiling of Salvia miltiorrhiza for MAO A and iNOS inhibitory activities. , 2004, Planta medica.

[31]  E. Sigel,et al.  The relative amount of cRNA coding for γ2 subunits affects stimulation by benzodiazepines in GABAA receptors expressed in Xenopus oocytes , 2002, Neuropharmacology.

[32]  E A Barnard,et al.  International Union of Pharmacology. XV. Subtypes of gamma-aminobutyric acidA receptors: classification on the basis of subunit structure and receptor function. , 1998, Pharmacological reviews.